Evaluation of insulin resistance in two kinds of South American camelids: llamas and alpacas.

Insulin resistance was evaluated in South American camelids, llamas and alpacas, by use of the minimal model test and the insulin tolerance test. Animals were catheterized for long-term studies and tamed to minimize stress during evaluation. Results indicated a low insulin sensitivity index (SI) = 0 to 0.97, median = 0.39 x 10(-4) min/uIU x ml, about a fifth the value in other mammals and humans. The KITT was between 1.43 and 3.19 %/min, also significantly lower than that reported for humans. Glycosylated hemoglobin concentration was 6%, and HbAlc concentration was 5.5%; red blood cell lifetime, as measured by use of the 51Cr method, was 120 days, similar to the value in humans. We concluded that llamas and alpacas have naturally higher blood glucose concentration than do humans and other mammals during the glucose tolerance test. Using the same mathematical tools to evaluate glucose metabolism as those used in people, South American camelids appear to be resistant to insulin. Thus, the South American camelid may be a useful new animal model for the study of sugar metabolism and various facets of diabetes mellitus, especially protection from the deleterious effects of glycosylation.

Protein-drug complexes important for immunoregulation and organ transplantation.

Recently, two structures of the Ser/Thr phosphorylase calcineurin in complex with FK506 and its cognate immunophilin, FKBP12, have been reported, both solved by small pharmaceutical companies focused on structure-based drug design. A realization, however, that the toxicities associated with calcineurin-mediated immunosuppressants might be mechanism based has driven the current interest in alternative approaches to autoimmunity prophylaxis and preventing transplant rejection. Regulatory approval in 1995 of the immunosuppressant prodrug mycophenolate mofetil, whose active metabolite, mycophenolic acid, inhibits inosine monophosphate dehydrogenase, has focused attention on the potential significance of the de novo purine-biosynthesis pathway as a target for immunosuppressive drugs, leading ultimately to the solution of enzyme structure as a drug design target. As this and other clinically relevant targets are discovered, elaborated and refined via the activity of their cognate agents (as was the case for the phosphatase calcineurin via the activity of cyclosporin), a critical opportunity should ensue for structural biology to exert a profound effect on the future development of these therapies.

Structure comparison of native and mutant human recombinant FKBP12 complexes with the immunosuppressant drug FK506 (tacrolimus).

The consequences of site-directed mutagenesis experiments are often anticipated by empirical rules regarding the expected effects of a given amino acid substitution. Here, we examine the effects of "conservative" and "nonconservative" substitutions on the X-ray crystal structures of human recombinant FKBP12 mutants in complex with the immunosuppressant drug FK506 (tacrolimus). R42K and R42I mutant complexes show 110-fold and 180-fold decreased calcineurin (CN) inhibition, respectively, versus the native complex, yet retain full peptidyl prolyl isomerase (PPIase) activity, FK506 binding, and FK506-mediated PPIase inhibition. Interestingly, the structure of the R42I mutant complex is better conserved than that of the R42K mutant complex when compared to the native complex structure, within both the FKBP12 protein and FK506 ligand regions of the complexes, and with respect to temperature factors and RMS coordinate differences. This is due to compensatory interactions mediated by two newly ordered water molecules in the R42I complex structure, molecules that act as surrogates for the missing arginine guanidino nitrogens of R42. The absence of such surrogate solvent interactions in the R42K complex leads to some disorder in the so-called "40s loop" that encompasses the substituent. One rationalization proposed for the observed loss in CN inhibition in these R42 mutant complexes invokes indirect effects leading to a misorientation of FKBP12 and FK506 structural elements that normally interact with calcineurin. Our results with the structure of the R42I complex in particular suggest that the observed loss of CN inhibition might also be explained by the loss of a specific R42-mediated interaction with CN that cannot be mimicked effectively by the solvent molecules that otherwise stabilize the conformation of the 40s loop in that structure.

X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex.

The X-ray structure of the ternary complex of a calcineurin A fragment, calcineurin B, FKBP12, and the immunosuppressant drug FK506 (also known as tacrolimus) has been determined at 2.5 A resolution, providing a description of how FK506 functions at the atomic level. In the structure, the FKBP12-FK506 binary complex does not contact the phosphatase active site on calcineurin A that is more than 10 A removed. Instead, FKBP12-FK506 is so positioned that it can inhibit the dephosphorylation of its macromolecular substrates by physically hindering their approach to the active site. The ternary complex described here represents the three-dimensional structure of a Ser/Thr protein phosphatase and provides a structural basis for understanding calcineurin inhibition by FKBP12-FK506.

Comparative X-ray structures of the major binding protein for the immunosuppressant FK506 (tacrolimus) in unliganded form and in complex with FK506 and rapamycin.

FK506 (tacrolimus) is a natural product now approved in the US and Japan for organ transplantation. FK506, in complex with its 12 kDa cytosolic receptor (FKBP12), is a potent agonist of immunosuppression through the inhibition of the phosphatase activity of calcineurin. Rapamycin (sirolimus), which is itself an immunosuppressant by a different mechanism, completes with FK506 for binding to FKBP12 and thereby acts as an antagonist of calcineurin inhibition. We have solved the X-ray structure of unliganded FKBP12 and of FKBP12 in complex with FK506 and with rapamycin; these structures show localized differences in conformation and mobility in those regions of the protein that are known, by site-directed mutagenesis, to be involved in calcineurin inhibition. A comparison of 16 additional X-ray structures of FKBP12 in complex with FKBP12-binding ligands, where those structures were determined from different crystal forms with distinct packing arrangements, lends significance to the observed structural variability and suggests that it represents an intrinsic functional characteristic of the protein. Similar differences have been observed for FKBP12 before, but were considered artifacts of crystal-packing interactions. We suggest that immunosuppressive ligands express their differential effects in part by modulating the conformation of FKBP12, in agreement with mutagenesis experiments on the protein, and not simply through differences in the ligand structures themselves.

Design, synthesis and structure of non-macrocyclic inhibitors of FKBP12, the major binding protein for the immunosuppressant FK506.

We have synthesized a series of non-macrocyclic ligands to FKBP12 that are comparable in binding potency and peptidyl prolyl isomerase (PPIase) inhibition to FK506 itself. We have also solved the structure of one of these ligands in complex with FKBP12, and have compared that structure to the FK506-FKBP12 complex. Consistent with the observed inhibitory equipotency of these compounds, we observe a strong similarity in the conformation of the two ligands in the region of the protein that mediates PPIase activity. Our compounds, however, are not immunosuppressive. In the FKBP12-FK506 complex, a significant portion of the FK506 ligand, its 'effector domain', projects beyond the envelope of the binding protein in a manner that is suggestive of a potential interaction with a second protein, the calcium-dependent phosphatase, calcineurin, whose inhibition by the FKBP 12-FK506 complex interrupts the T-cell activation events leading to immunosuppression. In contrast, our compounds bind within the surface envelope of FKBP12, and induce significant changes in the structure of the FKBP12 protein which may also affect calcineurin binding indirectly.

Structure and mechanism of interleukin-1 beta converting enzyme.

Interleukin-1 beta converting enzyme (ICE) processes an inactive precursor to the proinflammatory cytokine, interleukin-1 beta, and may regulate programmed cell death in neuronal cells. The high-resolution structure of human ICE in complex with an inhibitor has been determined by X-ray diffraction. The structure confirms the relationship between human ICE and cell-death proteins in other organisms. The active site spans both the 10 and 20K subunits, which associate to form a tetramer, suggesting a mechanism for ICE autoactivation.

Enthalpy of hydrogen bond formation in a protein-ligand binding reaction.

Parallel measurements of the thermodynamics (free-energy, enthalpy, entropy and heat-capacity changes) of ligand binding to FK506 binding protein (FKBP-12) in H2O and D2O have been performed in an effort to probe the energetic contributions of single protein-ligand hydrogen bonds formed in the binding reactions. Changing tyrosine-82 to phenylalanine in FKBP-12 abolishes protein-ligand hydrogen bond interactions in the FKBP-12 complexes with tacrolimus or rapamycin and leads to a large apparent enthalpic stabilization of binding in both H2O and D2O. High-resolution crystallographic analysis reveals that two water molecules bound to the tyrosine-82 hydroxyl group in unliganded FKBP-12 are displaced upon formation of the protein-ligand complexes. A thermodynamic analysis is presented that suggests that the removal of polar atoms from water contributes a highly unfavorable enthalpy change to the formation of C=O...HO hydrogen bonds as they occur in the processes of protein folding and ligand binding. Despite the less favorable enthalpy change, the entropic advantage of displacing two water molecules upon binding leads to a slightly more favorable free-energy change of binding in the reactions with wild-type FKBP-12.

Preliminary analysis of multiple crystal forms of the bovine cyclophilin-cyclosporin A complex.

The immunosuppressant cyclosporin-A (CsA) has been crystallized in complex with the bovine form of its major receptor protein cyclophilin (CyP) in three different forms by the hanging-drop vapor diffusion method. A hexagonal crystal form (P6(1)22 or P6(5)22; a = b = 110 A, c = 440 A) diffracts to 4 A resolution. Orthorhombic (P2(1)2(1)2(1) or P2(1)2(1)2; a = 154.3 A, b = 163.3 A, c = 94.7 A), and monoclinic (P2(1); a = 70.0 A, b = 162.5 A, c = 94.7, beta = 100.0 degrees) forms diffract to 2.2 A resolution. Self-rotation function analysis of the orthorhombic and monoclinic forms shows 52 point group local symmetry for both. A previously reported tetragonal crystal form of the complex also shares this local symmetry, suggesting that the observed motif may pre-exist in solution.

Structure-based drug design: applications in immunopharmacology and immunosuppression.

Structure-based drug design (SBDD) combines the power of many scientific disciplines, such as X-ray crystallography, nuclear magnetic resonance, medicinal chemistry, molecular modeling, biology, enzymology and biochemistry, in a functional paradigm of drug development. The current strength of SBDD lies in parlaying enzyme inhibitors into drugs, but a variety of technological advances over the past few years now makes it possible to address complex biological targets, such as those regulating immunosuppression and immunoactivation. Manual Navia and Debra Peattie discuss the SBDD paradigm and consider several of its achievements and challenges in immunopharmacology, particularly as these apply to the design of novel, potent immunosuppressants.

Structure-based drug design: applications in immunopharmacology and immunosuppression.

Structure-based drug design (SBDD) combines the power of many scientific disciplines, such as X-ray crystallography, nuclear magnetic resonance, medicinal chemistry, molecular modeling, biology, enzymology and biochemistry, in a functional paradigm of drug development. The current strength of SBDD lies in parlaying enzyme inhibitors into drugs, but a variety of technological advances over the past few years now makes it possible to address complex biological targets, such as those regulating immunosuppression and immunoactivation. Manuel Navia and Debra Peattie discuss the SBDD paradigm and consider several of its achievements and challenges in immunopharmacology, particularly as these apply to the design of novel, potent immunosuppressants.

Inhibition of human leukocyte elastase. 2. Inhibition by substituted cephalosporin esters and amides.

A variety of 7 alpha-methoxycephalosporin ester and amide sulfones were prepared and tested to determine the structure-activity relations for inhibition of human leukocyte elastase (HLE), a serine protease which has been implicated in several degenerative lung and tissue diseases. The most potent IC50 values were obtained with neutral, lipophilic derivatives, with the esters being more active than the amides. However, the best time-dependent inhibition in this series was observed with the p- and m-carboxybenzyl esters 7b and 7c. These results are discussed in terms of the proposed mechanism of inhibition as well as a molecular modeling study using the recently solved X-ray crystal structure of HLE.

A role for the aspartyl protease from the human immunodeficiency virus type 1 (HIV-1) in the orchestration of virus assembly.

Functional HIV-1 protease (PR) is required for the maturation of viral proteins, for the appearance of characteristic structural features in the virion (as determined by electron microscopy), and for the final assembly of mature virus. Most importantly, HIV-1 PR activity is required for the development of infectivity. Still largely undefined, however, is the timing and control of protease action in this assembly process. Based on the three-dimensional structure of HIV-1 PR2,3 and experimental data reported in the literature, we propose a comprehensive virus assembly model that highlights the role of HIV-1 PR, suggests further experiments to verify the validity of the model, and poses specific questions relevant to the ultimate exploitation of HIV-1 protease as a therapeutic target.

Crystallization of the aspartylprotease from the human immunodeficiency virus, HIV-1.

The aspartylprotease of the human immunodeficiency virus HIV-1 (NY5) has been crystallized in a form suitable for x-ray diffraction analysis. The crystals are tetragonal bipyramids and produce an x-ray diffraction pattern that exhibits the symmetry associated with space group P4(1)2(1)2 (or its enantiomorph, P4(3)2(1)2). The unit cell parameters are a = b = 50.3 A, c = 106.8 A, alpha = beta = gamma = 90 degrees; measurable diffraction intensities are observed to a resolution of 2.5 A. Density measurements indicate one molecule of 9,400 daltons/asymmetric unit. The symmetry of this space group could accommodate the proposed active dimer species of the protease if the 2-fold axis were coincident with one of the crystallographic 2-fold axes.

Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1.

The crystal structure of the protease of the human immunodeficiency virus type (HIV-1), which releases structural proteins and enzymes from viral polyprotein products, has been determined to 3 A resolution. Large regions of the protease dimer, including the active site, have structural homology to the family of microbial aspartyl proteases. The structure suggests a mechanism for the autoproteolytic release of protease and a role in the control of virus maturation.

Structure of human neutrophil elastase in complex with a peptide chloromethyl ketone inhibitor at 1.84-A resolution.

Human neutrophil elastase (HNE) has been implicated as a major contributor to tissue destruction in various disease states, including emphysema. The structure of HNE, at neutral pH, in complex with methoxysuccinyl-Ala-Ala-Pro-Ala chloromethyl ketone (MSACK), has been solved and refined to an R factor of 16.4% at 1.84-A resolution. Results are consistent with the currently accepted mechanism of peptide chloromethyl ketone inhibition of serine proteases, in that MSACK cross-links the catalytic residues His-57 and Ser-195. The structure of the HNE-MSACK complex is compared with that of porcine pancreatic elastase in complex with L-647,957, a beta-lactam inhibitor of both elastases. The distribution of positively charged residues on HNE is highly asymmetric and may play a role in its specific association with the underlying negatively charged proteoglycan matrix of the neutrophil granules in which the enzyme is stored.

Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels.

Charybdotoxin (ChTX), a protein present in the venom of the scorpion Leiurus quinquestriatus var. hebraeus, has been purified to homogeneity by a combination of ion-exchange and reversed-phase chromatography. Polyacrylamide gel electrophoresis, amino acid analysis, and complete amino acid sequence determination of the pure protein reveal that it consists of a single polypeptide chain of 4.3 kDa. Purified ChTX is a potent and selective inhibitor of the approximately 220-pS Ca2+-activated K+ channel present in GH3 anterior pituitary cells and primary bovine aortic smooth muscle cells. The toxin reversibly blocks channel activity by interacting at the external pore of the channel protein with an apparent Kd of 2.1 nM. The primary structure of ChTX is similar to a number of neurotoxins of diverse origin, which suggests that ChTX is a member of a superfamily of proteins that modify ion-channel activities. On the basis of this similarity, the three-dimensional structure of ChTX has been modeled from the known crystal structure of alpha-bungarotoxin. These studies indicate that ChTX is useful as a probe of Ca2+-activated K+-channel function and suggest that the proposed tertiary structure of ChTX may provide insight into the mechanism of channel block.